Microbiological desulfurization of gases

ABSTRACT

There is disclosed a method for desulfurizing gases by microbiological techniques which involve the use of chemoautotrophic bacteria of the Thiobacillus genus to convert sulfides to sulfates either as a sulfide removal process or as a process for producing biomass. More specifically, the invention involves the use of Thiobacillus denitrificans under aerobic conditions to oxidize sulfur compounds such as hydrogen sulfide to sulfate compounds. The process may be carried out by various techniques such as in a continuous bioreactor system using an immobilization matrix. The method is particularly suited to the disposal of hydrogen sulfide which has been otherwise removed from natural gas and producing a biomass byproduct.

BACKGROUND OF THE INVENTION

This invention relates to the desulfurization of gases and, inparticular, relates to the microbiological disposal of hydrogen sulfidewhich has been otherwise removed from natural gas.

Natural gas from a well may contain a number of undesirable componentswhich must be reduced to acceptable levels prior to distribution andsale. One of the most common problems in the gas industry is the removaland disposal of hydrogen sulfide. Hydrogen sulfide is an acid gas whichis toxic and quite corrosive in the presence of water. Natural gasdestined for the fuel market ordinarily must contain no more than 0.25grains per 100 standard cubic feet or 4 ppm on a volume basis.

The most commercially important treatment system for the removal anddisposal of hydrogen sulfide from natural gas consists of a combinationof the amine process for removal from the gas stream followed by theClaus process for sulfur recovery. In the amine process, aftercontacting the gas stream, the amine solvent is heated to 200°-300° F.to liberate H₂ S and regenerate the solvent which is recycled. It isimportant to note that the H₂ S is removed from the gas stream but thatit still must be disposed of. Hydrogen sulfide liberated duringregeneration of the amine solvent is converted to elemental sulfur bythe Claus process. In the Claus process, one third of the H₂ S of theacid gas stream received from the amine unit is burned with astoichiometric amount of air to produce sulfur dioxide according toEquation (1). If the entire acid gas stream is fed to the reactionfurnace, some conversion of H₂ S to elemental sulfur occurs in thefurnace according to Equation (2). Further conversion is achieved bypassing the reaction gas through a series of catalytic reactors whereelemental sulfur formation proceeds more toward completion at lowertemperatures. Alternately, one third of the acid gas stream may be fedto the reaction furnace for complete combustion of H₂ S to SO₂. The SO₂is then mixed with the remaining acid gases and fed to the catalyticreactors.

    H.sub.2 S+3/2 O.sub.2 →SO.sub.2 +H.sub.2 O+heat     (1)

    2H.sub.2 S+SO.sub.2 ⃡3S+2H.sub.2 O+heat        (2)

The Claus process produces a high quality elemental sulfur product andsalvage heat value as process credits which have a significant positiveimpact on the economics of the process. However, there are inherentlimitations and operating problems which may adversely affect theeconomics of the application of the process to H₂ S disposal. Theseinclude the following:

(1) The maximum conversion efficiency with as many as three catalyticreactors in series is only 96-97%. Further treatment of the Claus tailgas may be required to meet local air quality standards.

(2) Conversion efficiency is sensitive to variations in theconcentration of H₂ S in the acid gas feed stream.

(3) In the presence of carbon dioxide (CO₂) and light hydrocarbons, sidereactions can result in the formation of carbonyl sulfide (COS) andcarbon disulfide (CS₂) in the reaction furnace. The presence of COS andCS₂ may increase the number of catalytic stages requires for adequate H₂S conversion since COS and CS₂ hydrolysis requires higher temperaturesthan those which favor conversion of H₂ S to elemental sulfur accordingto Equation (2).

(4) At H₂ S concentrations of less than 40% the temperature of thereaction furnace is insufficient to result in complete combustion ofentrained hydrocarbons in the acid gas stream. Hydrocarbon reactionproducts can result in deactivation of the catalyst.

(5) Combustion of H₂ S in the reaction furnace becomes more unstablewith decreasing concentration of H₂ S in the acid gas feed stream. Atvery low H₂ S concentrations (less than 20%) preheating of air and acidgas streams is required. In addition SO₂ must be generated by burningrecycled elemental sulfur to ensure a proper stoichiometric H₂ S/SO₂ratio in the feed to the catalytic reactors.

With sufficient H₂ S available, a Claus plant can be profitable andoffset other costs associated with natural gas treatment with sulfursales and recovery of heat values. The break even point is influenced bythose factors discussed above. However, because of increasinglystringent air quality standards for sulfur emissions, the Claus processhas been applied in many treating situations where it is not economical.A need clearly exists for a new more economical technology in thesesituations especially with regard to acid gas streams with lowconcentrations of H₂ S. A new technology which featured a saleablebyproduct and greater conversion efficiency could also conceivablydisplace the Claus process in treating situations where it is presentlyregarded as economical. (Reference: Kohl, Arthur L. and Fred C.Riesenfeld, Gas Purification, Gulf Publishing Co., Houston, Tex., 3rdEd., p. 410-421 (1979)).

MICROBIAL REMOVAL OF HYDROGEN SULFIDE FROM A GAS

A number of microbial processes for the oxidation of H₂ S have beendescribed in the foreign patent literature. Those describing watertreatment are generally based on the innoculation of wastewaters withThiobacillus thioparus or other unspecified sulfur bacteria followed byaeration. (Polish Patent No. 98,513, Czechoslovakian Patent No. 178,012,U.S.S.R. Patent No. 1,070,120 and Polish Patent No. 106,991). T.thioparus has also been used to remove H₂ S from a gas which is bubbledthrough the culture (U.S.S.R. Patent No. 986,469). Mixed cultures ofbacteria from the Beggiatoa and Thiothrix genera have been utilized in asimilar manner (Japanese Patent No. 57,170,181). Thiobacillusferroxidans has been used as the basis of two gas treatment processes inwhich H₂ S is first precipitated as CuS or FeS. The sulfide precipitantis subsequently oxidized by the organism regenerating the precipitatingagent (West German Patent No. 3,300,402 and Japanese Patent No.58,152,488). All of these processes are aerobic. The latter two requirea very low, corrosion inducing pH.

A microbial process for the removal of H₂ S from a gas stream based onthe photosynthetic bacterium Chlorobium thiosulfatophilum has beenproposed as an alternative to the Claus or Stretford process. (Cork, D.J., "Acid Gas Bioconversion--An Alternative to the Claus Process," Dev.Ind. Micro., 23, 379-387 (1982); Cork, D. J. and S. Ma., "Acid GasBioconversion Favors Sulfur Production," Biotech. and Bioeng. Symp. No.12, 285-290 (1982); and Cork, D. J., R. Garunas and A. Sajjad,"Chlorobium limicola forma thiosulfatophilum: Biocatalyst in theProduction of Sulfur and Organic Carbon from a Gas Stream Containing H₂S and CO₂," Appl. and Env. Micro., 45, 913-918 (1983)). The processconverts H₂ S into a mixture of elemental sulfur and sulfate and claimssulfur and biomass as process credits. However, the requirement forradiant energy is a severe economic disadvantage whether suppliedartificially or collected from sunlight.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method fordesulfurizing gases by microbiological techniques. More particularly,the invention involves the use of chemoautotrophic bacteria of theThiobacillus genus to convert sulfides to sulfates either as a sulfideremoval process or as a process for producing biomass. Morespecifically, the invention involves the use of Thiobacillusdenitrificans under essentially aerobic conditions to oxidize sulfurcompounds such as hydrogen sulfide to sulfate compounds. A particularembodiment of the invention includes the use of specific strains ofThiobacillus denitrificans which will withstand high sulfideconcentrations and be resistant to a common biocide. The process of theinvention may be carried out by various techniques such as in acontinuous bioreactor system. The invention is particularly applicableto the disposal of H₂ S which has been otherwise removed from naturalgas and producing a biomass byproduct.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the preferred embodiment of the invention.

FIG. 2 is a graph showing the effect of temperature on the growth ofThiobacillus denitrificans.

FIG. 3 is a graph showing the viability of Thiobacillus denitrificans infree suspension in liquid medium without an energy source.

DESCRIPTION OF PREFERRED EMBODIMENTS Introduction

With the exception of photosynthetic organisms, the majority of thebiological world derives energy from the oxidation of organic compounds.However, there exists a group of microorganisms, predominantly bacteria,which may derive metabolic energy and reducing equivalents forbiosynthesis from the oxidation of inorganic elements and compounds.These microorganisms may also derive carbon for biosynthesis from aninorganic source such as carbon dioxide. This is termed achemoautotrophic mode of metabolism. The present invention employs suchbacteria and such mode of metabolism in order to remove sulfides fromgas streams.

A microbial gas desulfurization process offers several advantages whichcould make the process commercially viable. These include the following:

1. Direct conversion of hydrogen sulfide to sulfate is possible with norequirement for secondary sulfur recovery.

2. The energy requirements are low since the process operates at ambientor near ambient temperatures.

3. The nutrient is predominantly inexpensive mineral salts resulting ina low cost for chemicals.

4. The pH is moderate so that there are minimal corrosion problems.

5. No hazardous wastes are generated and there are minimal disposalproblems.

6. The process produces a high protein biomass and a sulfate salt whichcould represent salable products.

The ideal microorganism upon which to base a microbial hydrogen sulfideremoval process must possess several characteristics in addition to theability to oxidize hydrogen sulfide. The ideal microorganism would havesimple nutritional requirements in order to minimize chemical costs.Preferably the organism would be a strict autotroph, that is, theorganism would be capable of deriving all of its metabolic needs frominorganic sources. The ideal organism would also be capable of hydrogensulfide oxidation in an anaerobic as well as an aerobic environment togive greater versatility to the process. Preferably, the ideal organismwould produce a soluble oxidation product from the hydrogen sulfide inorder to facilitate separation of the oxidation product from thebiomass. The ideal organism would also exhibit a small size and simplemorphology so that it can be easily maintained in suspension. Manymicroorganisms produce an extracellular slime layer or capsid which cancause the microorganisms to adhere to walls and to each other. The idealorganism for hydrogen sulfide removal applications would not produce acapsid in order to prevent problems in transport of the organism. Auseful organism would also be able to withstand high pressures andmoderately high temperatures. An optimal pH near neutral would bedesirable in order to minimize corrosion. And, of course, the idealmicroorganism would also exhibit a high rate of hydrogen sulfideoxidation per unit biomass.

Many chemolithotrophic bacteria are capable of utilizing the oxidationof elemental sulfur and reduced or partially reduced sulfur compounds asa source of energy and reducing equivalents. However, taking intoconsideration the above factors, the bacterium Thiobacillusdenitrificans has been discovered to be uniquely suitable for theobjects of the present invention.

Thiobacillus denitrificans

Thiobacillus denitrificans (T. denitrificans) was first isolated in 1904by innoculation of an aqueous medium containing MgCl₂, K₂ HPO₄, KNO₃,Na₂ CO₃ and a sediment of elementary sulfur and CaCO₃ with canal wateror mud. A bacterial flora developed which oxidized the sulfur to sulfateand simultaneously reduced nitrate to elemental nitrogen. This was thefirst evidence of the existence of a chemolithotropic bacterium whichcould survive in the absence of oxygen. It was subsequently shown thatthiosulfate could be substituted for elemental sulfur. It was laterdemonstrated that a reduced nitrogen source was required for growth andT. denitrificans was cultivated in a defined medium for the first time.(Baalsrud, K. and K. S. Baalsrud, "Studies on Thiobacillusdenitrificans," Arch. Mikro., 20, 34-62 (1954)). This achievement led tothe first thorough study of the growth characteristics of the bacterium.These same authors reported the following:

(1) T. denitrificans is a facultative anaerobe utilizing oxygen underaerobic conditions or nitrate under anaerobic conditions as terminalelectron acceptor.

(2) T. denitrificans is an obligatory autotroph; that is, it cannotderive its metabolic needs from organic sources but is strictlydependent upon elemental sulfur and reduced sulfur compounds as energysources and carbon dioxide as a carbon source.

(3) Nitrate cannot serve as a sole source of nitrogen. Ammonia nitrogenis required for growth.

(4) Iron is required for growth. Good growth was reported in mediacontaining 0.25-8.3 micrograms Fe/ml.

(5) The optimum pH for growth of T. denitrificans is in the range of6.2-7.0. The organism is rapidly deactivated below pH 6.0.

Although it has been amply demonstrated that thiosulfate and elementalsulfur may be utilized as energy sources with oxidation to sulfate, theutilization of sulfide as an energy source by T. denitrificans, as wellas other Thiobacilli, has been the subject of some controversy in thepast. Some investigators have reported that cultures of T. denitrificansprovided with sulfide, usually supplied as Na₂ S, as the sole energysource failed to show an increase in protein content or sulfateconcentration in batch reactors. Others have observed the oxidation ofsulfide by whole cells or cell free extracts of T. denitrificans andother Thiobacilli. The deposition of elemental sulfur in growingcultures has been observed causing some investigators to speculate thatsulfide was oxidized to elemental sulfur and thiosulfate purelychemically and that these products were the true substrates for theThiobacilli. It is now apparent that those investigators who reportedthat T. denitrificans was incapable of growth on sulfide as an energysource came to an erroneous conclusion due to the very high initialsulfide concentrations used in their experiments (5-8 mM). Solublesulfide is toxic to Thiobacilli, as well as other microorganisms, inelevated concentrations. It has been demonstrated that T. denitrificanswill grow anaerobically on sulfide (Na₂ S) as an energy source ifsulfide is used as the growth limiting factor in a chemostat.(Timmer-ten Hoor, A., "Energetic Aspects of the Metabolism of ReducedSulphur Compounds in Thiobacillus denitrificans," Antonie vanLeeuwenhoek, 42, 483-492 (1976)). Under these conditions, theconcentration of sulfide in the culture is maintained at very low levelsand sulfide is oxidized to sulfate. Although this work established theability of T. denitrificans to utilize sulfide as an energy source underanaerobic and sulfide limiting conditions, growth on H₂ S under aerobicconditions had not been demonstrated prior to this work.

Growth and Maintenance of Cultures

The routine maintenance of T. denitrificans for stock cultures in amedium containing sulfide as an energy source would require continuousor semi-continuous addition of sulfide in such a way that sulfide didnot accumulate to inhibitory levels in the culture but sufficientsubstrate was made available for growth. Although this could be donewithin the scope of the present invention, the obvious difficultiesassociated with routine day-to-day maintenance of cultures in a sulfidemedium can be avoided by use of a non-toxic substrate, preferablythiosulfate. A typical thiosulfate maintenance medium is given by Tables1 to 3.

                  TABLE 1                                                         ______________________________________                                        Maintenance Medium                                                            Component         per liter                                                   ______________________________________                                        Na.sub.2 HPO.sub.4                                                                              1.2         g                                               KH.sub.2 PO.sub.4 1.8         g                                               MgSO.sub.4.7H.sub.2 O                                                                           0.4         g                                               NH.sub.4 Cl       0.5         g                                               CaCl.sub.2        0.03        g                                               MnSO.sub.4        0.02        g                                               FeCl.sub.3        0.02        g                                               NaHCO.sub.3       1.0         g                                               KNO.sub.3         5.0         g                                               Na.sub.2 S.sub.2 O.sub.3                                                                        10.0        g                                               Heavy metal solution                                                                            15.0        ml                                              Mineral water     50.0        ml                                              ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Heavy Metal Solution                                                          Component             per liter                                               ______________________________________                                        EDTA (Ethylenediaminetetraacetic                                                                    1.5      g                                              acid)                                                                         ZnSO.sub.4.7H.sub.2 O 0.1      g                                              Trace element solution                                                                              6.0      ml                                             ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Trace Element Solution                                                        Component       per liter                                                     ______________________________________                                        AlCl.sub.3.6H.sub.2 O                                                                         0.51         g                                                KI              0.14         g                                                KBr             0.14         g                                                LiCl            0.14         g                                                H.sub.3 BO.sub.3                                                                              3.06         g                                                ZnCl.sub.2      0.28         g                                                CuCl.sub.2.2H.sub.2 O                                                                         0.33         g                                                NiCl.sub.2.6H.sub.2 O                                                                         0.51         g                                                CoCl.sub.2.6H.sub.2 O                                                                         0.51         g                                                SnCl.sub.2.2H.sub.2 O                                                                         0.14         g                                                BaCl.sub.2.2H.sub.2 O                                                                         0.16         g                                                Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                 0.16         g                                                CuSeO.sub.4.5H.sub.2 O                                                                        0.14         g                                                NaVO.sub.3      0.024        g                                                ______________________________________                                    

The thiosulfate in the maintenance medium is the energy source, nitrateis the terminal electron acceptor allowing growth in the absence ofoxygen, bicarbonate is the carbon source and ammonium is the nitrogensource. The medium also includes a phosphate buffer and sources ofvarious essential mineral nutrients. This maintenance medium is similarto the S-8 medium for Thiobacilli recommended by the American TypeCulture Collection except that ammonium chloride has been substitutedfor ammonium sulfate as the source of reduced nitrogen with an increasein the concentration of ammonium ion, the concentrations of sodiumbicarbonate and hydrated magnesium sulfate have been increased and aknown source of trace elements has been added.

Oxidation of Hydrogen Sulfide

To produce a culture of T. denitrificans to be utilized for the removalof H₂ S from a gas, the organism is typically grown aerobically in thethiosulfate maintenance medium without nitrate at 30° C. and a pH of 7.0to an optical density at a wavelength of 460 nanometers (OD₄₆₀) ofapproximately 1.0. This optical density corresponds to greater than 10⁹cells per ml. As has previously been indicated, the purpose of thiscultivation on thiosulfate is to develop a sufficient concentration ofbiomass so that hydrogen sulfide can be fed to the reactor at anappreciable rate without exceeding the bio-oxidation capabilities of thebiomass. Otherwise, sulfide accumulates in the culture. During growth onthiosulfate an aeration rate of 200 to 300 ml/min/l of culture is used.It is advisable to supplement the air feed with 5% CO₂ to ensurecontinuous availability of a carbon source.

The pathways for sulfide and thiosulfate oxidation to sulfate in T.denitrificans are not independent but have two common intermediates. Inthe presence of thiosulfate the rate of sulfide oxidation is reducedbecause of competition for enzymes of the sulfur pathway. Therefore,there should be no residual thiosulfate in the culture when H₂ S isintroduced. This is readily accomplished by cultivating the cells to thepoint that all thiosulfate has been metabolized. The yield of T.denitrificans biomass on thiosulfate as an energy source has beenobserved to average 6.7 g dry wt./mole in batch reactors. The desiredconcentration of biomass can be developed by adjusting the thiosulfateconcentration in the medium with the precaution that the medium bethiosulfate limiting. When thiosulfate is depleted, H₂ S may beintroduced into the reactor at loadings of 8-10 mmoles/hr/g dry wt. ofbiomass. The culture must be sufficiently aerated that the reaction doesnot become oxygen limiting. Oxygen limitation has been observed at bulkoxygen concentrations below approximately 25 μM.

When H₂ S is introduced to a culture of T. denitrificans previouslygrown on thiosulfate, the H₂ S is immediately metabolized with noapparent lag. Under sulfide limiting conditions, less than 0.001 mM oftotal sulfide can be detected in the reactor medium. Provided then thatthe feed gas exits the reactor in equilibrium with the medium, very lowlevels of H₂ S in the outlet gas can be achieved (less than 1 ppmv).With 10,000 ppm H₂ S in the feed gas at one atmosphere, residence timesin the range of 1-2 sec are required if the average bubble diameter isapproximately 0.25 cm.

The introduction of H₂ S into a batch T. denitrificans reactor resultsin the accumulation of sulfate and biomass with a corresponding decreasein the ammonium concentration. No elemental sulfur accumulates in thereactor. The stoichiometry of the reaction in a batch reactor is givenby Table 4.

                  TABLE 4                                                         ______________________________________                                        Stoichiometry of Aerobic H.sub.2 S Oxidation by                               T. denitrificans in Batch Reactors.sup.a                                      ______________________________________                                        SO.sub.4.sup.-2 /H.sub.2 S                                                                    0.99 ± 0.05 mole/mole                                      O.sub.2 /H.sub.2 S                                                                            1.81 ± 0.11 mole/mole                                      NH.sub.4.sup.+ /H.sub.2 S                                                                     0.10 ± 0.02 mole/mole                                      OH.sup.- /H.sub.2 S                                                                           1.75 ± 0.16 equivalents/mole                               Biomass/H.sub.2 S                                                                             4.5 ± 0.9 grams/mole                                       ______________________________________                                         .sup.a 95% confidence intervals                                          

Certain aspects of the stoichiometry of any microbial process areaffected by the environment and the growth rate of the microbial cells.During batch growth these parameters are constantly changing. In acontinuous stirred tank reactor (CSTR) where a fresh nutrient feed(maintenance medium minus nitrate and thiosulfate) is fed to the reactorat the same rate at which mixed liquor is removed from the reactor, andwhere there is complete mixing, the environment and growth rate are heldconstant. Each of these parameters is controlled by the dilution rate atwhich the reactor is operated. The dilution rate D is defined byEquation (3) where q is the volumetric flow rate of nutrient to thereactor and v is the culture volume.

    D=q/v                                                      (3)

The stoichiometry of aerobic oxidation of H₂ S by T. denitrificans in aCSTR at dilution rates of 0.053 hr⁻¹ and 0.030 hr⁻¹ is given in Table 5.The yield of biomass was expected to be greater at the higher dilutionrate since a greater fraction of substrate H₂ S would be expected tosupport biosynthesis at higher growth rates. This has been observed tobe the case under anaerobic conditions. However, biomass yield fromaerobic growth on H₂ S was nearly the same at the two dilution ratesinvestigated.

                  TABLE 5                                                         ______________________________________                                        Stoichiometry Of Aerobic H.sub.2 S Oxidation By                               T. denitrificans in Continuous Flow Reactors.sup.a                            Dilution                              Biomass/                                Rate   SO.sub.4.sup.-2 /H.sub.2 S                                                               NH.sub.4.sup.+ /H.sub.2 S                                                                OH.sup.- /H.sub.2 S                                                                    H.sub.2 S                               (hr.sup.-1)                                                                          (mole/mole)                                                                              (mole/mole)                                                                              (eq./mole)                                                                             (g/mole)                                ______________________________________                                        0.053  1.04 ± 0.06                                                                           0.12 ± 0.01                                                                           1.77 ± 0.23                                                                         7.9 ± 0.7                            0.030  1.06 ± 0.09                                                                           0.11.sup.b 2.38.sup.b                                                                             8.1 ± 2.0                            ______________________________________                                         .sup.a 95% confidence intervals                                               .sup.b average of two determinations                                     

It has been reported in the literature that oxygen acts as an inhibitingsubstrate for T. denitrificans while growing aerobically on thiosulfate.Highest yields of biomass have been observed at low steady state oxygenconcentrations in the culture medium. (Reference: Justin, P. and D.P.Kelly, "Metabolic Changes in Thiobacillus denitrificans Accompanying theTransition from Aerobic to Anaerobic Growth in Continuous ChemostateGrowth", J. Gen. Micro., 107, 131-137 (1978). As shown in Table 6,experiments have not shown oxygen concentration in the range of 45-150μM to have a discernable effect on biomass yield for aerobic growth ofT. denitrificans on H₂ S.

                  TABLE 6                                                         ______________________________________                                        Biomass Yield As A Function of Steady State                                   Oxygen Concentration and Dilution Rate                                        Dilution Rate [O.sub.2 ]                                                                            Yield                                                   (hr.sup.-1)   (μM) (g Biomass/mole H.sub.2 S)                              ______________________________________                                        0.053          45     8.4                                                                    60     7.7                                                                   150     7.3                                                                   130     7.6                                                                   150     8.5                                                     0.030          90     7.7                                                                   120     9.0                                                                   100     7.5                                                     ______________________________________                                    

Indications of Upset and Recovery from Upset Conditions

Since H₂ S is an inhibitory substrate, it is imperative that the H₂ Sfeed rate to a T. denitrificans reactor not exceed the maximum capacityof the biomass for H₂ S oxidation. If the H₂ S oxidation capacity of thebiomass is exceeded, sulfide will accumulate in the reactor medium andinhibit the complete oxidation of H₂ S. Reactor upset is first indicatedby an increase in the optical density of the culture due to elementalsulfur accumulation. The culture takes on a whitish appearance. This isfollowed by H₂ S breakthrough. The upset condition is reversible ifexposure to the accumulated sulfide is not more than 2 to 3 hours.Reduction in H₂ S feed rate following an upset condition will reduce theH₂ S concentration in the outlet gas to pre-upset levels. In addition,elemental sulfur which accumulated during upset will be oxidized tosulfate upon reduction in H₂ S feed rate. The duration of the upsetcondition dictates the amount of reduction in the feed rate required forrecovery. The more sustained the period of upset, the more reduction infeed rate required. The maximum loading of a T. denitrificans culturewill be somewhat dependent upon both the metabolic state (growth rate)of the biomass and the environment of the biomass. Maximum loadings inthe range of 16 to 20 mmoles H₂ S/hr/g dry wt. biomass may be expectedunder aerobic conditions.

Effect of Heterotrophic Contamination

The medium described by Tables 1-3 will not support the growth ofheterotrophic microorganisms since there is no organic carbon source.However, if aseptic conditions are not maintained in the operation of aT. denitrificans reactor, heterotrophic contamination will develop inthe reactor. T. denitrificans releases organic material into the mediumin the normal course of growth or through lysis of nonviable cells. Thisorganic material then supports the growth of heterotrophs. In a T.denitrificans CSTR operated nonaseptically, the concentration ofheterotrophic contaminants will level off and remain constant after atime. The steady state concentration of contaminant is not surprisinglydependent upon the concentration of T. denitrificans. Contaminant levelsof up to 10% of the T. denitrificans concentration can be expected.Although the presence of a heterotrophic contamination can affect theend use of the biomass product of the process, the contamination doesnot affect H₂ S oxidation by T. denitrificans.

Characterization of Thiobacillus denitrificans

Other factors pertinent to the operation of a microbial gasdesulfurization process include the effects temperature and pressure,the toxicity of other sulfur compounds which may contaminate the feedgas, the effects of accumulating sulfate on cell activity and the effecton viability of maintenance in liquid culture in the absence of anenergy source.

Each of these parameters was examined under conditions in which T.denitrificans could be most easily cultured on a small scale, namelyanaerobically in thiosulfate maintenance medium. Conclusions reachedunder these conditions are likely to be pertinent to aerobic growth onH₂ S. As indicated previously, T. denitrificans cells grown onthiosulfate will readily oxidize H₂ S with no lag. It has also beenobserved that T. denitrifications cells growing on H₂ S can be switchedback and forth between aerobic to anaerobic conditions with no apparentlag in either direction. These results indicate that under any of thesegrowth conditions the cells contain basically the same complement ofenzymes.

The optimum temperature for growth of T. denitrificans has been reportedas 30° C. However, a temperature profile indicating relative growthrates above and below this optimum has not been published. A temperatureprofile is necessary to predict the effects of a temperature excursionor temperature gradients on overall and local growth rates in a culture.This can be especially important in the case of an inhibitory substratewhere a general or localized decrease in growth rate could result inaccumulation of the substrate to toxic concentrations. A temperatureprofile for T. denitrificans ATCC 23642 growing anaerobically onthiosulfate is given in FIG. 2 which indicates optimal growth over arelatively narrow range of temperatures with complete inhibition ofgrowth above 40° C. However, viable counts have shown that attemperatures as high as 45° C., no measurable effect on viability isobserved for exposures of up to 5 hours.

Growth of T. denitrificans in thiosulfate medium at 30° C. at elevatedpressures indicates that total pressure has no significant effect ongrowth at pressures of up to 1800 psig N₂ or 1000 psig CH₄. Theseresults are shown in Table 7. Viability was demonstrated at theconclusion of each test by growth on thiosulfate agar and noheterotropic contamination was indicated in that no growth appeared onnutrient agar. In a microbial gas desulfurization process themicroorganisms may be subjected to rapid pressurization-depressurizationcycles. Table 8 summarizes the results of rapidpressurization-depressurization at 1250 psig of N₂ on viable count in aculture of T. denitrificans originally grown at that pressure onthiosulfate. Table 8 indicates that repeatedpressurization-depressurization has no significant effect on viability.

                  TABLE 7                                                         ______________________________________                                        Effect of Pressure on Growth of T. denitrificans                              on Thiosulfate in Liquid Culture                                                                                   Optical                                            Press.           Incubation                                                                              Density                                  Culture   (psig)    Gas    Time (days)                                                                             (460 nm)                                 ______________________________________                                        TEST      400       N.sub.2                                                                              3         1.10                                     CONTROL   0                3         1.14                                     TEST      600       N.sub.2                                                                              4         1.05                                     CONTROL   0                4         1.20                                     TEST      750       N.sub.2                                                                              3         0.75                                     CONTROL   0                3         1.20                                     TEST      1000      N.sub.2                                                                              3         0.75                                     CONTROL   0                3         1.00                                     TEST      1240      N.sub.2                                                                              3         0.75                                     CONTROL   0                3         0.83                                     TEST      1800      N.sub.2                                                                              3         1.08                                     CONTROL   0                3         0.87                                     TEST      500       CH.sub.4                                                                             3         0.85                                     CONTROL   0                3         0.80                                     TEST      1000      CH.sub.4                                                                             3         1.20                                     CONTROL   0                3         0.62                                     ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Effect of Sequential Pressurization-Depressurization                          Cycles at 1250 psig N.sub.2 on Viability of                                   T. denitrificans in Liquid Culture                                            Pressurization/                                                               Depressurization                                                                              Viable Count                                                  Cycles          (cells/ml)                                                    ______________________________________                                        0               5.2 × 10.sup.8                                          1               3.9 × 10.sup.8                                          2               4.2 × 10.sup.8                                          3               3.4 × 10.sup.8                                          4               4.3 × 10.sup.8                                          ______________________________________                                    

Various sulfur compounds common to natural gas are somewhat toxic to T.denitrificans. Those compounds are methyl mercaptan (CH₃ SH), carbondisulfide (CS₂), carbonyl sulfide (COS) and dimethyl sulfide (CH₃ SCH₃).The order of toxicity to wild type T. denitrificans is CH₃ SH>CS₂ >COS,CH₃ SCH₃. All are toxic at a partial pressure of 200 mmHg. At partialpressures sufficiently low to be tolerated none are metabolized.

As H₂ S is oxidized by T. denitrificans, a sulfate salt accumulates inthe medium. Under aerobic conditions, the counter ion of the sulfate inthis salt will be determined by the counter ion of the hydroxideequivalents added to the culture to maintain pH. For example, if KOH isthe alkali used for pH control, the oxidation product of H₂ S isprimarily present as K₂ SO₄. Whether the reactor is operated batchwiseor on a continuous basis, the concentration of sulfate salt will bedependent upon the rate of H₂ S oxidation per unit volume of culture.The tolerance of T. denitrificans for the accumulating sulfate salt,therefore, has a major influence on the operation of the reactor. Wildtype T. denitrificans is tolerant of up to 450 nM K₂ SO₄ when grownanaerobically on H₂ S. Above approximately 500 mM, incomplete oxidationof H₂ S is observed with the accumulation of elemental sulfur andproduction of N₂ O from incomplete reduction of nitrate. The organism isless tolerant of Na₂ SO₄ ; however, normal reactor operation is observedat Na₂ SO₄ concentrations of 300-400 mM. (NH₄)₂ SO₄ causes incomplete H₂S oxidation at concentrations above 150-200 mM.

As noted above, another factor pertinent to the operation of a microbialgas desulfurization process is the effect on viability of maintenance inliquid culture in the absence of an energy source as would occur if thefeed gas to the process were shut off for a period of time. Asillustrated by FIG. 3, the viable count in a culture of T. denitrificansdecreases with time in the absence of an energy source. However, if aworking culture contains at least 10⁹ cells/ml, a sufficient number ofviable cells will exist after as much as 20 days to provide an adequateinnoculum to restart the process if care is taken not to overload thebiomass.

Another factor which will influence the economics of a microbial gasdesulfurization process is the value of the biomass produced. Theprotein content of T. denitrificans whole cells grown on H₂ S is 60%±3%by dry weight. This protein content is intermediate between that ofsoybeammeal (51%) and fish meal. (72%), the two most commerciallyimportant sources of bulk protein. The quality of a bulk protein sourceas a food supplement is dependent not only upon the protein content butalso upon the amino acid composition of that protein. Table 9 gives theamino acid composition of T. denitrificans whole cell protein when theorganism is grown on H₂ S. Table 10 compares the amino acid compositionof T. denitrificans whole cell protein, with respect to the tenessential amino acids in a mammalian diet, to that of soybeam meal andfish meal Table 10 indicates that T. denitrificans whole cell protein,on a g/100 g basis, contains more of nine of these amino acids thansoybeam meal. The only possible exception is tryptophan which has notbeen determined for T. denitrificans protein. Fish meal contains greaterquantities of isoleucine, lysine, threonine and possibly tryptophan. Thecysteine content of T. denitrificans is so low as to be undetectable.Also pertinent to the nutritional quality of the biomass is the mineralcontent. A trace element analysis of T. denitrificans biomass grown onH₂ S is given in Table 11.

                  TABLE 9                                                         ______________________________________                                        Amino Acid Composition of T. denitrificans                                    Whole Cell Protein                                                            Amino Acid         g/100 g Protein                                            ______________________________________                                        Alanine            7.8                                                        Arginine           7.3                                                        Aspartic Acid + Asparagine                                                                       10.3                                                       Glutamic Acid + Glutamine                                                                        11.1                                                       Glycine            5.2                                                        Histidine          5.5                                                        Isoleucine         5.4                                                        Leucine            9.7                                                        Lysine             7.1                                                        Methionine         3.7                                                        Phenylalanine      4.4                                                        Proline            4.4                                                        Serine             3.4                                                        Threonine          4.4                                                        Tyrosine           3.7                                                        Valine             6.7                                                        ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Essential Amino Acid Content of                                               T denitrificans Protein Compared to                                           Soybean Meal and Fish Mean Proteins                                                  g/100 g Protein                                                        Amino Acid                                                                             Soybean Meal Fish Meal T. denitrificans                              ______________________________________                                        Arginine 6.2          6.8       7.3                                           Histidine                                                                              2.1          2.8       5.5                                           Isolencine                                                                             4.9          6.3       5.4                                           Leucine  6.6          9.4       9.6                                           Lysine   5.6          9.4       7.1                                           Methionine                                                                             1.2          3.5       3.7                                           Phenylalanine                                                                          4.3          4.3       4.4                                           Threonine                                                                              3.3          4.7       4.4                                           Tryptophan                                                                             1.2          1.1       --                                            Valine   4.7          6.5       6.7                                           ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Trace Element Analysis of T. denitrificans                                    Whole Cells Grown on H.sub.2 S                                                             ppm (wt)                                                         ______________________________________                                        Fe             7530                                                           Zn              140                                                           Mg             5800                                                           Cu              90                                                            Ca             3550                                                           Mn             1710                                                           Na             3330                                                           K              1670                                                           Total Ash       12%                                                           Total Sulfur   0.9%                                                           ______________________________________                                    

Mutant Strains

The present invention includes not only the use of wild strains of T.denitrificans such as ATCC 23646 (American Type Culture Collection,Rockville, Md.), but also mutant strains. For example, sulfide tolerantstrains of T. denitrificans are desirable to make the proposed microbialgas desulfurization process more resistant to upset from excess H₂ Sfeed and possibly more tolerant of other sulfur compounds. A biocideresistant strain could provide a means of controlling heterotrophiccontamination and therefore produce a microbially pure biomass productwithout the expense of maintaining aseptic conditions by sterilizationof feed streams. Therefore, the term T. denitrificans as used herein andin the claims includes mutants thereof.

Continuous Flow Reactor with Biomass Recycle

A simple CSTR is an economically impractical reactor configuration withrespect to volumetric productivity for the proposed microbial gasdesulfurization process except where very small amounts of H₂ S areremoved. However, a completely mixed, homogeneous environment for thecells is required to avoid localized inhibitory concentrations ofsulfide. The most practical reactor configuration presently contemplatedfor a microbial gas desulfurization process based on T. denitrificans isa CSTR with biomass recycle. Recycle of the biomass allows much higherbiomass concentrations to be maintained in the reactor. In addition,with biomass recycle, the hydraulic retention time and biomass retentiontime are decoupled. Therefore, high dilution rates can be used toreplenish the culture medium and control the environment of the cells.Biomass concentration and the quality of the cells' environment will bethe two most important variables in maximizing volumetric productivitywhile maintaining reactor stability. With cell recycle, these twovariables are independently controlled.

For a CSTR with biomass recycle, the microbial cells must continuouslybe harvested from the reactor liquid waste stream. The more commonmethods of continuous harvesting of microbial cells include continuouscentrifugation and tangential flow filtration. An alternative toharvesting and recycle of free cell biomass is the use of an immobilizedbiomass which is the preferred embodiment of the present invention aswill be described hereinafter. If the immobilization matrix issufficiently dense, biomass from the reactor effluent may be harvestedby low gravity sedimentation. An immobilization matrix appropriate forgrowing cells must allow release of new cells into the surroundingmedium. Therefore, the reactor effluent will contain both immobilizedcells which could be readily recovered and recycled and free cells. Ifthe free cells are to represent a process credit, they must berecovered. Therefore, even when immobilized cells are utilized, a freecell recovery problem still exists. However, since these cells are notrecycled back to the reactor, treatment of the process stream (with aflocculating agent, for example) to improve sedimentation properties canbe tolerated.

It was noted previously that a facultative organism offers advantages inversatility in a microbial gas desulfurization process. One of theseadvantages is revealed in the use of a porous immobilization matrix forthe T. denitrificans biomass. Oxygen is only a sparingly soluble gas. At30° C. and at saturation with air at 1 atmosphere, the concentration ofoxygen in the culture medium characteristic of this process is on theorder of 200-250 μM. Therefore, the driving force for mass transfer ofO₂ into the immobilization matrix is relatively low. Therefore, in apurely aerobic system only the outermost fraction of the matrix volumemay be populated with metabolically active cells. Research has shownthat T. denitrificans will preferentially use oxygen as an oxidant inthe presence of NO₃ ⁻ ; however, NO₃ ⁻ is immediately utilized when O₂is depleted. The incorporation of nitrate in the culture medium atconcentrations of only a few mM would result in a much higher drivingforce for mass transfer of NO₃ ⁻ into the matrix than O₂. Therefore, inthe presence of a small concentration of NO₃ ⁻ the entire void volume ofthe immobilization matrix could be populated with metabolically activecells. The interior of the matrix would operate anaerobically while theexterior operates aerobically. This is hereafter referred to as a mixedaerobic/anaerobic system. The details of anaerobic metabolism of H₂ S inT. denitrificans have been described in a previous patent application(see U.S. Patent Application Ser. No. 787,219, filed Oct. 15, 1985).

The particular immobilization matrix does not form a part of the presentinvention and any known matrix material may be used which is suitablefor the T. denitrificans. By way of example only, see U.S. Pat. Nos.4,153,510 and 4,286,061. Also, any suitable procedure well known in theprior art for immobilizing T. denitrificans cells on the matrix materialcan be used in the present invention as long as the cells may grow anddivide while releasing new cells from the matrix.

Referring now to FIG. 1, the immobilized biomass is loaded into thereactor 10 which is filled with maintenance medium without thiosulfate.A limited amount of nitrate may be incorporated in the medium if a mixedaerobic/anaerobic metabolism is desired in the immobilization matrix.The hydrogen sulfide containing gas is passed into the reactor throughline 12 and the treated gas with the H₂ S removed flows out line 14. Airis introduced into the reactor through line 11. The maintenance mediumand immobilized biomass (the slurry) are stirred by the mixer 16 inorder to achieve homogeneity, avoid localized inhibitory concentrationsof sulfide and obtain good mass transfer.

Withdrawn from the reactor 10 is a slurry stream 18 which containspartially spent nutrient, free floating bacteria which have beenexpelled from the immobilizing support material and the dissolvedsulfate formed in the reactor during the H₂ S removal process. A form offiltration may be employed to prevent the immobilized biomass from beingwithdrawn from the reactor along with the slurry. Alternatively,immobilized biomass withdrawn from the reactor along with the slurry isremoved from the slurry in separator 20 and recycled to the reactorthrough line 22. This separator 20, for example, may be a conventionalsettling basin or hydrocyclone.

The slurry from the separator 20 is then passed to the settler 24through line 26 to perform the removal of the free cell bacterialbiomass. The preferred method of accomplishing this removal is byintroducing a flocculating agent as indicated at 28 into the slurry toassist in the agglomeration and settling of the bacterial biomass. Thebacterial biomass product is then removed from the settler as indicatedat 30. The remaining liquid from the settler 24 now contains the spentnutrient and the sulfate. This liquid is passed through line 32 into theevaporator/fractional crystallizer 34. In the evaporator/fractionalcrystallizer, the process is controlled depending upon the relativeconcentrations of the sulfate and the remaining nutrients in the liquidsuch that only the sulfate is crystallized or such that the crystallizedsulfate will contain only that amount of nutrient which has also beencrystallized which can be tolerated in the sulfate product dependingupon its intended end use. The product from the evaporator/ fractionalcrystallizer 34 is passed through line 36 to the separator 38 where thecrystals are separated from the remaining liquid. The separated crystalscontaining primarily the sulfate is removed from the settler 38 throughline 40. The liquid from the settler 38 contains primarily only theremaining nutrient materials which were present in the withdrawn spentnutrient. This liquid is recycled through line 42 to the reactor 10along with fresh nutrient introduced through line 44 to replenish thespent nutrient. As shown in FIG. 1, the system for practicing thisinvention includes a temperature controlling heat exchanger 46 in whichthe medium being fed into the reactor 10 is controlled to a temperatureof about 30° C. if wild type T. denitrificans is utilized or a highertemperature if a temperature tolerant strain is utilized. It may also beadvantageous to precondition the makeup medium being introduced throughline 44 to the optimal temperature. Further measures which can beemployed to control temperature is to precondition the entering gas 12to the optimal temperature, include a heat exchanger within the reactor10 and insulate reactor 10.

For the purpose of giving a specific example of the present invention,the treatment of 25×10⁶ standard cubic feed/day (7.08×10⁸ standardliters/day) of natural gas at 600 psig (42 atmospheres or 4238kilopascals absolute) containing 1.5 mol % H₂ S will be used. Theaerobic process of the present invention could not normally be used todirectly remove the H₂ S since the oxygen would contaminate the naturalgas. Therefore, a conventional amine plant would be used to treat thenatural gas and remove the H₂ S along with CO₂. The amine plant wouldremove 1.87×10⁴ gram-moles of H₂ S per hour. This H₂ S plus anyaccompanying CO₂ removed represents the feed stream 12 to the reactor10. If a stable loading (sulfide limiting conditions) of 10.0 millimolsH₂ S per hour per gram biomass is assumed, then 1.87×10⁶ g of T.denitrificans biomass will be required to treat the gas stream. With asuitable choice of immobilization matrix, immobilized whole cellreactors can be operated with 40% slurries of porous immobilizationbeads with low rates of attrition. Furthermore, the beads can developinternal populations of viable cells which pack the beads to 50% oftheir theoretical packing density. However, to be conservative, a 20%slurry of 200 micron beads with a maximum packing density of 25% oftheoretical is assumed. This small bead diameter is selected to minimizeinternal mass transfer resistances. If a T. denitrificans cell isidealized as a cylinder with a diameter of 0.5 micron and length of 1.5microns, a 200 micron bead would contain 1.67×10⁷ cells at maximumpacking. A 20% slurry of beads would therefore contain 1.33×10¹⁴ cellsper liter. A viable cell density of 10⁹ cells per milliliter is roughlyequivalent to 0.5 grams dry weight of biomass per liter. Therefore, a20% slurry of 200 micron beads with maximum cell packing would contain67 grams per liter of immobilized cells. To be conservative, 50 gramsper liter is chosen as a design basis. Therefore, if the free cellbiomass is neglected, a total bubble free culture volume of 3.7×10⁴liters will be required to treat the gas stream described above.

The economics of a microbial gas desulfurization process are obviouslystrongly influenced by the volumetric productivity of the bioreactor. Asecond important factor is the dilution rate at which the reactor isoperated. Process economics are favored by lower dilution rates. Thereactor effluent must be processed to recover biomass and the sulfatesalt, both of which may be taken as a process credit. Lower dilutionrates result in a lower rate of flow of the effluent stream andincreased concentrations of free cell biomass and sulfate which reduceprocessing costs. In addition, lower dilution rates decrease pumpingcosts. However, the reduction of dilution rate to improve processeconomics has a limitation dictated by the tolerance of the biomass forthe accumulating sulfate salt in the culture medium. The maximumconcentration of the sulfate salt which can be tolerated withoutsignificant inhibition of growth, and therefore H₂ S oxidation, willdetermine the minimum dilution rate at which the reactor can beoperated. As noted above, some control can be exerted by choice of thesulfate counter ion which is determined primarily by the hydroxidecounter ion in the pH adjusting solution.

I claim:
 1. A method for treating a gas stream containing hydrogensulfide to remove said hydrogen sulfide comprising the steps of:a.culturing T. denitrificans in the absence of H₂ S in a maintenancemedium containing a quantity of thiosulfate to produce a desiredpopulation of T.denitrificans, thereby reducing said quantity ofthiosulfate and forming a slurry of T. denitrificans in maintenancemedium, b. removing any thiosulfate which may remain in said slurry, c.flowing said gas stream and oxygen through said slurry in a reactorwhereby said T. denitridicans removes said hydrogen sulfide underaerobic conditions and produces sulfate and whereby said population ofT. denitrificans increases to produce excess T. denitrificans, the ratioof gas stream flow to the quantity of T. denitrificans being such thatthe average maximum loading of H₂ S is no greater than 20 millimoles ofH₂ S per hour per gram dry weight of T. denitrificans and the quantityof O₂ is maintained at a level of at least 25 μM, d. removing a portionof said slurry from said reactor, said slurry containing excess T.denitrificans and sulfate, e. removing at least a portion of said excessT. denitrificans and at least a portion of said sulfate from saidremoved slurry thereby leaving a remaining slurry, and f. recycling saidremaining slurry back to said reactor.
 2. A method for treating a gasstream containing hydrogen sulfide to remove said hydrogen sulfidecomprising the steps of:a. culturing T. denitrificans in the absence ofH₂ S in a maintenance medium containing a quantity of thiosulfate toproduce a desired population of T. denitrificans, and to reduce saidquantity of thiosulfate, b. removing any thiosulfate which may remain,c. immobilizing said population of T. denitrificans in immobilizationmatrices, d. introducing said population of T. denitrificans in saidimmobilization matrices into a reactor containing maintenance mediumfree of thiosulfate thus creating a slurry, e. flowing said gas streamand oxygen through said slurry in said reactor whereby said T.denitrificans removes said hydrogen sulfide under aerobic conditions andproduces sulfate and whereby said population of T. denitrificansincreases to produce excess T. denitrificans which are released fromsaid immobilization matrices, the ratio of gas stream flow to thequantity of T. denitrificans being such that the average maximum loadingof H₂ S is no greater than 20 millimoles of H₂ S per hour per gram dryweight of T. denitrificans and the quantity of O₂ is maintained at alevel of at least 25 μM, f. removing a portion of said slurry from saidreactor, said removed slurry containing a portion of said immobilizationmatrices containing T. denitrificans and a portion of said excess T.denitrificans which have been released from said immobilization matricesand a portion of said produced sulfate and a portion of said maintenancemedium, g. removing from said slurry and recycling to said reactor saidimmobilization matrices containing said T. denitrificans and producing aremaining slurry, h. removing said remaining slurry said excess T.denitrificans to produce a T. denitrificans biomass product and a spentmaintenance medium solution containing sulfate, and i. removing at leasta portion of said sulfate from said spent maintenance medium solutionand recycling said spent maintenance medium solution back to saidreactor.
 3. A method as recited in claim 2 and further including thestep of introducing fresh maintenance medium to said reactor.
 4. Amethod as recited in claim 2 wherein the temperature in said reactor iscontrolled to about 30° C.
 5. A method as recited in claim 4 and furtherincluding the step of introducing fresh maintenance medium to saidreactor.
 6. A method as recited in claim 2 wherein said maintenancemedium includes nitrate whereby said T. denitrificans on the outermostportion of said immobilization matrices will function aerobically andthe T. denitrificans on the innermost portions of said matrices willfunction anaerobically through the metabolism of nitrate.
 7. A method asrecited in claim 3 wherein said maintenance medium includes nitratewhereby said T. denitrificans on the outermost portion of saidimmobilization matrices will function aerobically and the T.denitrificans on the innermost portions of said matrices will functionanaerobically through the metabolism of nitrate.
 8. A method as recitedin claim 4 wherein said maintenance medium includes nitrate whereby saidT. denitrificans on the outermost portion of said immobilizationmatrices will function aerobically and the T. denitrificans on theinnermost portions of said matrices will function anaerobically throughthe metabolism of nitrate.
 9. A method as recited in claim 5 whereinsaid maintenance medium includes nitrate whereby said T. denitrificanson the outermost portion of said immobilization matrices will functionaerobically and the T. denitrificans on the innermost portions of saidmatrices will function anaerobically through the metabolism of nitrate.10. A method as recited in claim 3 wherein said maintenance mediumfurther contains nitrate as a terminal electron acceptor, bicarbonate asa carbon source and ammonium as the nitrogen source.
 11. A method asrecited in claim 2 wherein hydroxide is added to said slurry for pHcontrol between 6.2 and 7.0.
 12. A method as recited in claim 2 whereinsaid gas stream and oxygen flow through said slurry in step (e) furthercontains up to 5% CO₂ to supplement the carbon supply.